PROJECT SUMMARY Epithelial-stromal interactions are critical determinants of epithelial cell behavior in a tumor. While the stromal microenvironment and alterations in the stromal extracellular matrix (ECM), influences invasive and migratory properties at the invasive front, the role of the epithelial matrix in influencing cancer cell behavior are less well understood. Extracellular matrix (ECM) perturbations in epithelial cancers contribute to metastatic spread accounting for 80% of the cancer-related deaths worldwide. The molecular cues that affect the ECM and precede stromal influences on epithelial cell metastasis, is a critical first step at identifying pathways that can be molecularly targeted in the future. The overarching goal of this project is to determine whether alterations in the extracellular matrix of cancer epithelial cells can sufficiently alter cancer cell invasion, migration and metastatic propensity. Fibronectin (FN) matrix dysregulation in response to hypoxia using hypoxia mimetics and 1% O2 will be investigated in the MCF10A progression series to determine whether cellular responses in the three cell lines alternate between premalignant and malignant phenotypes or preinvasive and invasive phenotypes. During low O2 levels in the cells, Hypoxia-inducible factor (HIF)-1? and HIF2? proteins undergo stabilization and translocate to the nucleus to enable transcription of genes involved in cell survival, angiogenesis, and metabolism. An angiogenic transcriptional target of HIF? is VEGF (Vascular endothelial growth factor), which interacts with the C-terminal Heparin-II domain of the FN protein. Using non-contacting and contacting transwell assays, with endothelial cells, the role of FN fibrillogenesis in binding VEGF or releasing VEGF to promote angiogenesis will be determined. While breast cell lines are used in this study, the project goal applies to epithelial cancer cell invasion and migration and hypoxia- mediated angiogenesis that depend on FN fibrillogenesis. The long-term goal of this study is to provide new information on unfavorable FN matrix changes that can be perhaps stabilized that in combination with existing therapeutic strategies will help alleviate cancer cell aggression.